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PHA(eng)
1. UNIVERSITA’ DEGLI STUDI DI CATANIA
FACOLTA’ DI FARMACIA
PhD in Medicinal Chemistry
GIUSEPPE PUZZO
BACTERIAL FERMENTATION AND MICROWAVE-
ASSISTED SYNTHESIS FOR THE PRODUCTION OF
BIODEGRADABLE AND BIOCOMPATIBLE
POLYMERS USABLE IN THE PHARMACEUTICAL
FIELD.
Coordinator: Tutor:
Prof. Giuseppe Ronsisvalle. Prof. Alberto Ballistreri.
Ciclo XXIV
4. Polyhydroxyalkanoates (PHA)
P
Poly(3-hydroxyalkanoates)
with R= alkyl or functional group
n
PHASCL: short-chain length PHA
3-5 carbon atoms
PHAMCL: medium-chain length PHA
6-14 carbon atoms
5. Physical and chemical properties.
•Average molecular weight ranging between 5·104 and1·106 Da
•Enantiomerically pure
•Biodegradable and biocompatible
Extension at
Polymer T
Tg(°C) T
Tm (°C) C
Cristallinity (%)
b
break (%)
P
P(3HB) 15 175 50-80 5
P(3HB-co-3HV) -1 145 56 50
P(3HB-co-4HB) -7 150 43 444
PP -15 176 50 400
6. Applications of PHAs in medicine and pharmaceuticals.
•Sutures. •Bone graft substitutes.
•Temporary heart valves. •Carrier for drug delivery.
8. The aim of the thesis
Explore new strategies for obtaining new polymers which, in the
pharmaceutical field, have feature of biodegradability and
biocompatibility with wider opportunity of utilization with respect to
poly(3-hydroxybutyrate) (PHB) by:
1. The study on the capabilty to P. aeruginosa to grow and
synthesize PHAs from Long Chain Fatty Acids (LCFA) or
vegetable oils, with better yields or with new structures and new
properties.
2. Chemical synthesis of new coplymers and terpolymers by
transesterification reaction microwave assisted.
18. Assuming a Bernoullian (random) distribution of repeating units
in these copolymers, the probability of finding a given Ax, By…
Nz can be calculated by the Leibnitz formula as follows:
A measure of the fit of the calculated oligomers intensities to
the experimental ones is given by the agreement factor (AF);
the lower AF, the closer fit.
∑(I + I
expi
. calci
. )2
AF= i
∑I 2
expi
.
i
19. R R
R CH CH CO [O CH CH2 CO ]
n
O CH CH2 COOH
Negative ion ESI mass spectrum of the partial pyrolisate of the PHA from
enicosanoic acid. R may be an un n-etyl, n-butyl, n-hexyl, n-octyl, n-
decyl and n-dodecyl group.
21. Brassica carinata
production’s seeds
Remaining
Oil De-oiling flour
Modified As such As such Formulation
Lubrificants Fertilizer Soil products
Lubricants
Energy products
Biofuels
Agricoltural
oils
22. Table 4. PHA production from P. aeruginosa cultured on
differents substrates.
Substrate Dry cell weight PHA content PHA yield
(mg/L) (% dry cell weight) (mg/L)
B. Carinata oil 1000 5,0 50
Oleico acid 380 15,0 57
Erucic acid 2 866 9,3 81
Nervonic acid 416
416 10
10.0 42
29. T:3 T:3
12 T:3 8, 11
T:1 D:1 T:2 Δ:1 T:1
6 T:2
6 T:2 6
8 9 D:1 5
O:1 Δ:1 8
6 O:1
6 7 7
T:3 T:2 5
T:3
9 5
5
138 136 134 132 130 128 126 124 122 120
(ppm)
C-NMR spectra of the PHA obtained from B. carinata oil in the region
13
of the olefinc signals.
30. Chemical structure of the PHA from B. carinata oil. This PHA
is made up of all the repeating units constituting the PHA
from erucic acid, plus the unsatureted ones shown here.
O:1 D:1 Δ:1 T:2 T:3
3 2 1 3 2 1 3 2 1 3 2 1 3 2 1
O CH CH CO O CH CH CO O CH CH CO O CH CH CO O CH CH CO
2 n 2 o 2 p 2 r 2 s
4CH 4CH 4 CH 4 CH 4 CH
2 2 2 2 2
5CH 5CH 5 CH 5 CH 5 CH
2 2
6CH 6CH 6 CH 6 CH 6 CH
2
7CH 7CH 7 CH 7 CH 7 CH
2 2 2
8CH 8 CH 8 CH 8 CH 8 CH
3 2
9CH 9 CH 9 CH 9 CH
2 2
10 CH 10 CH 10CH 10CH
3 2 2 2
11CH 11 CH 11CH
2 2
12CH 12CH 12CH
3 2
13CH 13CH
2 2
14CH 14CH
3 3
31. Table 6. Physical characteristics of the PHAs isolated
from P. aeruginosa grown on B. carinata oil and on oleic,
erucic and nervonics acids.
Sustrate Tg (°C) Tm (°C) ΔHm (J/g) Mw x 10-3 Mw/Mn
B.carinata oil -47 - - 56 1,8
Oleico acid -52 - - 57 2,2
Erucic acid -46 50 16,1 122 1,9
Nervonic acid -43 50 15,5 114 2
32. Dimeri
R R
311 R CH CH CO [O CH CH2 CO ] n
O CH CH2 COOH
100 283
% Intensità 339
Trimeri
Tetrameri
60 453 481
255 367 425 509 595 623
393 535 567 651 679
20
300 400 500 600 700
x3 (m/z)
100
Pentameri
% Intensità
Esameri
765 Eptameri
60 737 793 907
879 935 1049
709 821 1077
851 963 1021 1105
991 1133
1161
20
700 800 900 1000 1100
(m/z)
Negative ion ESI mass spectrum of the partial pyrolisate of the PHA
from erucic acid. R may be a n-propyl, n-pentyl, n-heptyl, n-nonyl or n-
undecenyl group.
33. Table 7. Experimental and calculated relative amounts of
the partial pyrolisis products of the PHA produced by P.
aeruginosa from erucic acid.
m/z ESI Calculated
Dimers
C-O 255 10 9
C-D; O2 283 24 24
C-Δ; O-D 311 26 26
O-Δ; D2 339 18 19
O-T:1 365 6 7
D-Δ 367 8 7
D-T:1 393 6 4
Δ-T:1 421 2 2
Trimers
C22 397 4 4
C24 425 12 12
C26 453 20 19
C28 481 18 20
C30 :1 507 5 7
C30 509 13 13
C32:1 535 10 9
C32 537 6 5
C34:1 563 8 6
34. R R
Dimeri
R CH CH CO [O CH CH2 CO ]
n
O CH CH2 COOH
311
100
283
339 Trimeri
% Intensità
60 255 Tetrameri
453 481
367 425 509 623 651
393 535 567 595 679
20
300 400 (m/z) 500 600 700
100 x 4
Pentameri
737 765 793 Esameri
% Intensità
60 709 821 907 Eptameri
879 935 963
849
991 1021 1049 107711051133
20
700 800 900 (m/z) 1000 1100
Negative ion ESI mass spetrum of the partial pyrolisate of the PHA
from B. carinata. R may be a n-pentaenyl, n-heptaenyl, n-nonaenye, n-
undecadieyil or n-undecatrienyl group.
35. Design For Efficient Energy: Energy requirements should be recognized for their
environmental and economic impacts and should be minimized. Synthetic methods should be
conducted at ambient temperature and pressure.
Heating mechanisms heat exchange Heating with Microwave
Benefits:
Energy saving
Process Efficiency
Restrictions on the use of halogenated
solvents
36. What are the microwave
The microwaves are not ionizing electromagnetic waves having a
wavelength between 1 mm (ν = 300 GHz) and 1 m (ν = 300 MHz),
they are located in the area of the spectrum between the
frequencies of the infrared and the radio waves.
The frequency of 2.45 (± 0.05) GHz, corresponding in vacuum at a
wavelength (λ) of 12.2 cm, is that used for applications in the
domestic field, scientific, medical, and for many industrial
processes.
37. Chemical synthesis of copolyesters.
CH 3 O O
O CH CH 2 C + O CH 2 CH 2 CH2 CH2 CH 2 C
PHB n PCL m
1. PTSA·H2O, Chloroform, Toluene (reflux)
2. Azeotropic (dehydration)
CH 3 O O
O CH CH 2 C O CH 2 CH 2 CH 2 CH 2 CH 2 C
n m
P(HB-co-CL)
38. Table 8. Transesterification Conditions, Yields, Molecular
Weights, and Degree of Transesterification of P(HB-co-CL)
Copolymers.
Sample HB/CLa Yield (%) Mw·103 b Mw/Mn c DT d DR e RT(h) f
Conventional
heating
A 54/46 15 7.8 1,41 0,16 0,3 1/2
B 45/55 23 n.d. n.d. 0,21 0,52 2/2
C 75/25 19 n.d. n.d. 0,42 0,92 3/2
D 55/45 10 7.9 1,3 0,37 0,74 5/2
Microwave
heating
E 55/45 52 5.2 1,3 0,1 0,21 1/2
F 48/52 49 6.4 1,27 0,12 0,25 2/2
G 55/45 30 9 1,2 0,17 0,36 3/2
H 46/54 26 12 1,24 0,31 0,63 5/2
a
Molar composition of the resulting copolymers. b Weight-average molecular weight.
c
Molecular weight distribution. d Degree of transesterification at the end of the second
stage of the reaction. e Degree of randomness at the end of the second stage of the
reaction. f Duration in hours of the two transesterification stages. n.d.: not detemined.
39. a
O CH3 O
O CH2 CH2 CH 2 CH 2 CH2 C O CH CH2 C
e f g h i l m b c d n
a
e g
f+h
i
c
b
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
(ppm)
200 MHz 1H-NMR spectra of the copolymer P(HB–co-45%mol CL)
(sample D) obtained with conventional heating.
40. m’ n’ a
H HO O CH O
3
H3C S O CH CH2 CH 2 CH 2 CH2 C O CH CH2 C
n e 2 f g h i l m b c d n
H HO
m n
e
g
f+h
a
i
c
m+m’ n+n’ b
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
(ppm)
200 MHz 1H-NMR spectra of the copolymer P(HB–co-54%mol CL)
(sample H) obtained with microwave heating.
41. a
O CH3 O
O CH2 CH2 CH 2 CH 2 CH2 C O CH CH2 C
e f g h i l m b c d n
f g
e h
i
c
a
b
l d
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
(ppm)
50 MHz 13C-NMR spectra of the copolymer P(HB–co-45%mol CL)
(sample D) obtained with conventional heating.
42. m’ n’ a
H HO O CH O
3
H3C S O CH CH2 CH 2 CH 2 CH2 C O CH CH2 C
e 2 f g h i l m b c d n
H HO
m n h
g
e i f
c
a
l b
d m+m’ n+n’
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
(ppm)
50 MHz 13C-NMR spectra of the copolymer P(HB–co-54%mol CL)
(sample H) obtained with microwave heating.
43. CCC
BCC
BBC
BBB
BCB CBB
CCB
CBC
174.5 174.0 173.5 173.0 172.5 172.0 171.5 171.0 170.5 170.0 169.5 169.0 168.5
(ppm)
13
C-NMR spectral expansion of the carbonyl region of the copolymer
(sample H).
44. 2X B 2XC
LB = LC =
( I BC + I CB )
( I BC + I CB )
where XB and XC are the dyad mole fractions of HB and CL calculable by the
equations:
X B = I BB + 1 2 ( I BC + I CB ) X C = I CC + 1 2 ( I BC + I CB )
DT = I BC + I CB DR = 1 LB + 1 LC
For a random copolymer of 1:1 composition, these parameters are expected
to assume the values LB = LC = 2, DT = 0.5 and DR = 1.
45. : Spettro MALDI-TOF della frazione eluita dopo il massimo del tracciato GPC del copolimero P(HB-co- 45 mol%CL) (campione D).
5894
5810
5838
5754
5866
5782
5922
5950
1000
800
5750 5850 5950
600 (m/z)
400
200
4500 5000 5500 6000 6500 7000 7500
(m/z)
MALDI-TOF mass spectrum of the fraction eluting after the maximum
of the GPC trace of the copolimer P(HB-co- 45 mol%CL) (sample D).
46. : Spettro MALDI-TOF della frazione eluita dopo il massimo del tracciato GPC del copolimero P(HB-co- 45 mol%CL) (campione D).
Chemical synthesis of terpolyesters.
CH3
O CH O CH2 O
3
O CH2 CH2 CH2 CH2 CH2 C + O CH CH2 C O CH CH2 C
m n o
PCL P(HB-co-HV)
1. PTSA·H2O, Chloroform, Toluene (reflux)
2. Azeotropic (dehydration)
CH
3
O CH O CH2 O
3
O CH2 CH2 CH2 CH2 CH2 C O CH CH2 C O CH CH2 C
m n o
P(HB-co-HV-co-CL)
47. Table 9:Transesterification Conditions, Yields, Molecular
Weights, and Degree of Transesterification of P(HB-co-HV-
co-CL) Terpolymers.
Sample HB/HV/CLa Resa (%) Mw·103 b Mw/Mn c DT d DR e RT(h) f
Conventional
heating
L 51/15/34 30 6.7 1,36 0,61 1,05 1/2
M 47/12/41 19 11.3 1,16 0,71 1,41 2/2
N 48/13/39 13 8.1 1,12 0,81 1,54 3/2
Microwave
heating
P 62/14/24 51 8.1 1,3 0,64 1,64 1/2
Q 58/15/27 37.5 9.1 1.9 0,7 1,27 2/2
R 68/13/19 35 6.7 1,2 0,75 1,47 3/2
a
Molar composition of the resulting terpolymers. b Weight-average molecular weight.
c
Molecular weight distribution. d Degree of transesterification at the end of the second
stage of the reaction. e Degree of randomness at the end of the second stage of the
reaction. f Duration in hours of the two transesterification stages.
48. Spettro 1H-NMR a 200 MHz del terpolimero P(3HB-co-12%mol 3HV-co-41%mol CL) (campione M).
m
g CH
3
O CH O n CH O
3 2
O CH CH CH CH CH C O CH CH C O CH CH C
2 2 2 2 2 2 p 2 q
a b c d e f m h i l n o o
g+n
a b+d
i+p e
m
h+o c
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
(ppm)
200 MHz 1H-NMR spectra of the terpolymer P(HB-co-12%mol HV-
co-41%mol CL) (sample M).
49. m
x’ y’ CH
g 3
H HO O CH O n CH O
3 2
H3C S O CH CH CH CH CH C O CH CH C O CH CH C
a 2 b 2 c 2 d 2 e2 f m h i 2 l
n o p2 q o
H HO
x y
g+n
i+p b+d
a
e m
y+y’ x+x’ h+o
c
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
(ppm)
200 MHz 1H-NMR spectra of the terpolymer P(HB-co-15%mol HV-
co-27%mol CL) (sample Q).
50. m
CH3
g
O CH3 O n CH O
2
O CH CH2 CH2 CH CH2 C O CH CH C O CH CH C
2 2 2 l o p2 q o
a b c d e f m h i n
g
i
h
ed
a
b c
l+q
n m
f o p
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
(ppm)
50 MHz 13C-NMR of the terpolymer P(HB-co-12%mol HV-co-41%mol CL)
(sample M).
51. m
x’ y’ CH3
g
H HO O CH3 O
nCH O
2
H3C S O CH2 CH CH2 CH CH C O CH CH2 C O CH CH 2 C
a b2 c d2 e 2 f m h i l n o p q o
H H O
x y g
i
h
e
l+q a
b cd
m
o n
y+y’ x+x’ p
f
180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0
(ppm)
50 MHz 13C-NMR spectra of the terpolymer P(HB-co-15%mol HV-
co-27%mol CL) (sample Q).
52. Espansione dello spettro 13C NMR della regione dei carbonili del terpolimero M.
BB
BV,VB
CC
BC
BC
CV
VC VV
173.6 173.2 172.8 172.4 172.0 171.6 171.2 170.8 170.4 170.0 169.6 169.2 168.8
(ppm)
13C-NMR spectral expansion of the carbonyl region of the terpolymer
(sample M).
53. 2 XB 2 XV
LB = LV =
( I CB + I BC + I CV + I BV + IVB ) ( I CB + I BC + I CV + I BV + I VB )
2 XC
LC =
( I CB + I BC + I CV + I BV + I VB )
where XB, XV, and XC are the dyad mole fractions of HB, HV and CL calculable by
the equations:
X B = I BB + 1 2 ( I CB + I BC + I CV + I BV + I VB ) X V = I VV + 1 2 ( I CB + I BC + I CV + I BV + I VB )
X C = I CC + 1 2 ( I CB + I BC + I CV + I BV + I VB )
DR = 1 LB + 1 LC + 1 LV DT= ICB+IBC+ICV+IVC+IBV+IVB/2XB XC+2XCXV+2XBXV
54. Conclusion 1
Through bacterial fermentation were obtained for the first
time PHA using very long chain fatty acids (VLCFA), more
than 20 C atoms and B. carinataI oil. The PHA produced by
fatty acid with odd number of carbon atoms are flexible
materials whose physical characteristics do not vary
significantly as a function of the side chain, although longer
pendant groups confer a greater speed of recrystallization.
The PHA produced by using erucic and nervonic acids, are
transparent as well, partially crystalline and therefore they
show rubber-like characteristics. Their proposed use is as
scaffold in tissue engineering and in the pharmaceutical
delivery system.
The PHA from B. carinata oil is a transparent material, totally
amorphous. The presence of double bonds allows the
derivatization and functionalization.
55. Conclusion 2
By chemical synthesis were obtained biodegradable and
biocompatible copolymers and terpoIymers. The structure of
these polymers is random or microblock depending on the
duration of the reaction or the amount of catalyst used and
the type of heating used. At equal number of hours of
reaction, and degree of transesterification catalyst used, the
use of microwaves has allowed to obtain higher yields for
both copolymers that for the terpolymers.
Copolymers and terpolymers obtained by this method are
capable of producing micro-and nanoparticles used in the
drug delivery system.
Editor's Notes
Plastics are used almost in every aspect of human lives. Plastics are used as a container, yarn and fabrics, household, cord, etc. Recently, plastics are produced by petrochemical industries as a polyethylene, Polypropylene, polystyrene, poly vinyl chloride etc. The raw material of these plastic industries is derived from fossil fuel. There are two problem facing this plastic : The lack of new resources of feedstock (fossil fuel) The problem with their disposal since synthetic plastic is not biodegradable. Motivation: new route of bio-plastic Bioplastics is produced by microorganism The raw material is renewable sources such as corps. Bioplastic can be degraded in environment then converted by nature to the raw material again This cycle is harmless to the environment, i.e. no produce additional CO2 to the atmosphere Therefore this route is a sustainable system
Polyhidroxyalkanoates is a polyester – has a link between hydroxy and carboxyl units of monomers. The most PHA has hydroxyl unit at the third carbon If R is H -> P(3-hydroxypropanoate) If R is CH3 -> PHB If R is CH2-CH3 -> PHV Generally PHA divided into 2 groups: SCL = with 2-5 carbon atoms MCL = with 6-14 carbon atoms This variation gives different chemical and mechanical properties of polymers
There are two type of microorganism which are synthesizing PHA First group of bacteria synthesizes PHA in a limited non-carbon-nutrients condition. Therefore, to have high enough cell density, cultivation is carried out in adequate nutirient to let the cells grow until they reach high enough cell density. Thus, the nutrient is modified to stop the cell grow and let the cell to produce PHA polymers inside their body. This mechanism is belong to R eutropha, P. oleovorans Another group of bacteria synthesizes PHA in a growing phase, hence the nutrient should be controlled to supply the cells to build their body and also synthesize the polymer. This mechanism can be found in A. latus, A. vinelandii, rec. E. coli
The substrates of PHA synthesis are mainly glucose and alkanoate (acid). Glucose can derived from carbohydrates which are carried out from various corps such as corn, sugar cane, wheat, potato, tapioca etc. Alkanoate mostly come from fatty acid in form of vegetable oil and fats An interesting PHA synthesis substrate is a waste. For instance, a effluent of palm oil mill could be a good substrates for MCL-PHA since it contain a lot of fatty acid. By applying different kind of bacteria, the various PHA could be obtain from these wastes. (This will be discuss later)
